Cloning and Structure of XFGFRL1 cDNA
To isolate the cDNA fragment of XenopusFGFRL1/ndk, a Xenopus EST clone that showed high similarity to the human FGFRL1 cDNA sequence with an E-value of 1.1E-21 was selected using the TIGER Xenopus laevis Gene Index (XGI). A 241-bp fragment was amplified from a cDNA library of Xenopus stage 17/18 whole-embryo using primers constructed from the EST sequence. The deduced amino acid sequence encoded in this fragment shows 74% identity to that of human FGFRL1 in the second Ig-like domain. cDNA library screening was performed using the 241-bp fragment as a probe. An isolated cDNA clone (1,874 bp long) contained 310 bp of 5′ untranslated region (UTR), 1,452 bp of open reading frame (ORF) encoding 484 amino acid residues and 112 bp of 3′ UTR. This predicted protein is highly related to human FGFRL1 as well as planarian NDK. Unlike these proteins, which contain three FGFR-related Ig-like domains, planarian NDK does not have conserved cysteine residues in the third Ig-like domain or a typical transmembrane domain. Their cytoplasmic regions lack a kinase domain characteristic of FGF receptors and do not have significant homology to any other known sequences. We noticed that the reported C-terminal sequence of mouse FGFRL1 (Sleeman et al., 2001; Wiedemann and Trueb, 2001) does not match those of the human and Xenopus sequences and that an additional reading frame of the mouse sequence does match to the human and Xenopus sequences, suggesting that the reported mouse sequences need to be reexamined. Based on the similarity to the overall structure of human FGFRL1, the gene we isolated is likely to be the Xenopus ortholog of FGFRL1, referred to as XFGFRL1 or Xndk. Xenopus FGFRL1 shows 66% or 17% identity to that of human or planarian, respectively, over the entire region (Fig. 1).
Figure 1. Alignment of the deduced amino acid sequences of FGFRL1/NDK. Planarian, Xenopus, and human FGFRL1 are designated as NDK, XFGFRL1, and hFGFRL1, respectively. Boxed in black, identical residues in all of the three, or boxed in gray, in two of them; dotted underlines, signal peptides (only for XFGFRL1 and hFGFRL1); underlines, immunoglobulin (Ig) -like domains (the third Ig-like domain is present only in XFGFRL1 and hFGFRL1); box, transmembrane domains (only for XFGFRL1 and hFGFRL1). Accession numbers: NDK, AB071948; XFGFRL1, AB117614; hFGFRL1, AJ277437. FGFRL1, fibroblast growth factor receptor-like 1; X, Xenopus; h, human.
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Expression Patterns of XFGFRL1 in Early Development
The temporal expression pattern of XFGFRL1 in early Xenopus development was analyzed by reverse transcription-polymerase chain reaction (RT-PCR; Fig. 2). XFGFRL1 expression was first detectable at the early neurula stage (stage 13/14) and maintained at later stages. The spatial and temporal expression pattern was analyzed by whole-mount in situ hybridization. At the late gastrula stage (stage 12), XFGFRL1 expression was widely detected in the anterior region when cleared with benzyl benzoate/benzyl alcohol (Fig. 3A). At the early neurula stage, expression of XFGFRL1 became prominent in the anterior head region with a clear posterior boundary (Fig. 3B). To examine the expression domain precisely, stained embryos were sectioned and stained with DAPI (4′,6-diamidine-2-phenylidole) to distinguish germ layers by the differences in nuclear density. This examination by sectioning indicated that XFGFRL1 expression in the anterior region is localized to the prechordal plate, anterior endoderm, and archenteron roof (Fig. 3C) and also to anterior part of the paraxial and lateral mesoderm (Fig. 3D).
Figure 2. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of developmental expression of XFGFRL1/Xndk. Developmental stages are indicated above each lane. RT- is the negative control, and histone H4 was used as the loading control. XFGFRL1, Xenopus fibroblast growth factor receptor-like 1.
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Figure 3. Spatiotemporal expression of XFGFRL1/Xndk at late gastrula to neural tube stages visualized by whole-mount in situ hybridization. Hybridization probes used for XFGFRL1 and XFGF8 are as indicated in the pictures (XFGFRL1 in black; XFGF8 in blue). “cleared,” embryos were treated with benzyl benzoate/benzyl alcohol to become transparent. Numbers indicate developmental stages (Nieuwkoop and Faber, 1967). A: Lateral view of late gastrula. Anterior toward the left; dorsal is upward. B: Lateral view of early neurula. Anterior toward the left; dorsal is upward. C: Sagittal section of early neurula (stage 13/14). D: Transverse section of early neurula (stage 13/14). The section corresponds to the position shown by a line with d in B. E: Anterolateral (left) and lateral view (middle; anterior toward the left; dorsal is upward) of XFGFRL1 and XFGF8 (right) expression at neurula. Arrowhead, ventrolateral expression in a crescent shape. F,G: Horizontal (F) or transverse (G) section of neurula (stage 17/18). The planes of section are indicated by a line with f or g in E. Arrowheads in lower panels indicate that crescent-shaped expression (see E, right panel) is seen in the sensorial layer of epidermis. H: Sagittal section of neurula (stage 17/18). I: Lateral (left and middle panels; anterior toward the left; dorsal is upward) and anterior view (right panels; dorsal is upward) of neural tube stage embryos. Crescent-shaped expression (see E, right panel) is separated into two stripes as indicated by arrowheads (see I, lower panels). C,D,F–H: Sections of stained embryos are shown in brightfield (left) and DAPI (4′,6-diamidine-2-phenylidole) staining (right). XFGFRL1, Xenopus fibroblast growth factor receptor-like 1; XFGF8, Xenopus fibroblast growth factor-8; ae, anterior endoderm; anr, anterior neural ridge; ar, archenteron roof; bc, blastocoel; ey, eye; fb, forebrain; lp, lateral plate; mhb, midbrain-hindbrain boundary; no, notochord; op, otic placode; pcp, prechordal plate; pm, paraxial mesoderm; sm, somitic mesoderm. Scale bars = 200 μm in C,D,F–H.
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As XFGFRL1 is structurally related to FGFR and may modulate FGF signaling, as shown in the functional analysis of NDK in Xenopus embryos (Cebria et al., 2002), we observed that expression domains of XFGFRL1 at later stages resemble those of Xenopus, chick, and mouse FGF8 (Heikinheimo et al., 1994; Ohuchi et al., 1994; Crossley and Martin, 1995; Mahmood et al., 1995; Christen and Slack, 1997; Crossley et al., 2001) in the anterior neural ridge (ANR), midbrain–hindbrain boundary (MHB) region, forebrain, otic vesicles, somites, and visceral arches as described below. We therefore carried out whole-mount in situ hybridization for both XFGFRL1 and XFGF8 at stage 17/18 and later. As neurulation proceeded, the mesodermal expression of XFGFRL1 gene expanded to the posterior and ventral regions (Fig. 3E, left and middle panels), and additional expression was detected at the ANR (Fig. 3E, left panel), while expression of XFGF8 was detected at the ANR and also in the ventrolateral region in a crescent shape (Fig. 3E, right panel; indicated by an arrowhead). Section examination showed that the expression of both XFGFRL1 (Fig. 3F, upper panels) and XFGF8 (Fig. 3F,H, lower panels) at the ANR was detected primarily in its sensorial layer. While the ventrolateral crescent-shaped expression of XFGF8 is mainly in the anterior sensorial layer of the ectoderm (indicated by arrowheads in Fig. 3F,G, lower panels), XFGFRL1 is more broadly expressed in the mesoderm adjacent to the crescent-shaped XFGF8 expression domain (Fig. 3E, left; 3F,G, upper panels). The expression region of XFGFRL1 in the overall anterior head region is shown in a cleared embryo (Fig. 3E, middle panel) and includes the prechordal plate, anterior endoderm, and archenteron roof (Fig. 3G,H, upper panels), similar to the early neurula (Fig. 3B–D). At the neural tube stage (stage 22), XFGFRL1 expression was detected in the eye vesicles and the forebrain (Fig. 3I, upper left and right panels). In addition, the expression in the MHB region was manifested when stained embryos were cleared (Fig. 3I, upper middle panel). At this stage, XFGF8 expression was detected at the rostral midline of the forebrain, MHB, and otic placodes (Fig. 3I, lower panels), and the crescent-shaped expression was separated into two stripes (indicated by arrowheads in Fig. 3I, lower panels).
During the tail bud stages (stages 26 to 33/34), additional expression of XFGFRL1 was detected in the visceral arches and the otic vesicles (Fig. 4A,B, left panels). XFGFRL1 was coexpressed with XFGF8 in the MHB, forebrain, visceral arches, and somites at stage 26 and later, although the expression level of XFGF8 in the somites decreases at later stages (Fig. 4A–D,G,H). In the otic vesicles, XFGFRL1 and XFGF8 appeared to be expressed differently in the ventral and anterior region, respectively, of the vesicles (Fig. 4B; see below). XFGFRL1 expression was also detected in the pineal gland (Fig. 4B, left panel; 4C, left and middle panels; 4E, upper left panel), while XFGF8 expression was detected in the pronephros and pronephric duct at stage 26 (Fig. 4A, right panel), and probably at the boundary between prosomeres 2 and 3 at stages 26 to 33/34 (indicated by arrowheads in Fig. 4A,B, right panel) according to the observation in the chick embryo (Crossley et al., 2001).
Figure 4. Spatiotemporal expression of XFGFRL1/Xndk and XFGF8 at tail bud stages visualized by whole-mount in situ hybridization. The expression of XFGFRL1 is compared with that of XFGF8 as indicated (XFGFRL1 in black; XFGF8 in blue). “cleared,” embryos were treated with benzyl benzoate/benzyl alcohol to become transparent. A,B: Lateral view. Anterior toward the left; dorsal is upward. Arrowheads (right panels; see also C, right panel) indicate the region that probably corresponds to the boundary of prosomeres 2 and 3 in the diencephalon (see text). C–H: Comparison of XFGFRL1 (left or upper panel) and XFGF8 (right or lower panel) expression by horizontal or transverse (F, upper panel) sections of stage 33/34 (C–G) and stage 26 (H) embryos. C: Expression in the midbrain–hindbrain boundary. Middle panels are enlargement of left panel, showing that XFGFRL1 is expressed in the pineal gland. D: Forebrain expression. XFGF8 is also expressed in anterior head mesenchyme (right panel). E: Eye expression in whole embryos (left panels) and in horizontal sections (middle and right panels). Left panels show enlargement of the head region of the embryos in B. The sections correspond to the position shown by a line with e in left panels. White dotted lines show the outline of the eyes (upper left panel) and the ciliary margin and commissural plate (lower middle panel). White arrowheads show XFGF8 expression in the eye and commissural plate (lower left panel). F: Otic vesicle expression. XFGFRL1 (upper panel) is expressed in ventral and XFGF8 (lower panel) is expressed in the anterior part of the otic vesicles. G: Visceral arch expression. Middle and right panels show enlargement of left panels. Both XFGFRL1 and XFGF8 are expressed in the visceral pouches as indicated by arrows. Turquoise staining seen in the expression of XFGFRL1 was due to depletion of nitro blue tetrazolium inside of embryos by strong staining of the ectodermal region. Thus, XFGFRL1 expression (shown in blue and turquoise) is wider than that of XFGF8. H: Somite expression. Staining is localized to nuclear and perinuclear regions as indicated by arrows. XFGFRL1, Xenopus fibroblast growth factor receptor-like 1; XFGF8, Xenopus fibroblast growth factor-8; cm, ciliary margin; cp, commissural plate; ey, eye; fb, forebrain; le, lens epithelium; me, mesenchyme; mhb, midbrain–hindbrain boundary; ov, otic vesicle; pd, pronephric duct; pg, pineal gland; pn, pronephros; so, somite; tz, transitional zone; va, visceral arch; vp, visceral pouch. Scale bar = 100 μm.
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Horizontal sections showed that XFGFRL1 expression was detected in the overall forebrain, while XFGF8 expression was localized in the rostral midline of the forebrain and in anterior head mesenchyme (Fig. 4D) and that XFGFRL1 and XFGF8 were coexpressed in the visceral pouches of pharyngeal endoderm (Fig. 4G). Figure 4E shows that XFGFRL1 expression in the eye is localized in the lens epithelial cells, transitional zone, and ciliary margin (Fig. 4E, upper panels), while XFGF8 expression in the eye is localized in the anterior ciliary margin and commissural plate (Fig. 4E, lower panels) as has been reported in chick embryos (Crossley et al., 2001). The expression of XFGFRL1 and XFGF8 in ventral and anterior region, respectively, of the otic vesicles was confirmed by examination of a cross-section for XFGFRL1 (Fig. 4F, upper panel) and a horizontal section for XFGF8 (Fig. 4F, lower panel). Expression of both XFGFRL1 and XFGF8 in the somites at stage 26 was localized around the nucleus (Fig. 4H, indicated by arrows).